Project 5: Nanofluidics Devices for Rapid Single Cell Analysis of Protein Expression A critical problem in cancer biology stems from the inability to perform biochemical and molecular characterization at the single cell level. To address this issue, we propose to develop a point-of-care microfluidics platform that accepts "as collected" patient samples and within 30 min provides relevant biochemical information to the clinician. The microfluidics devices will include sample processing and a micro-flow cytometer coupled with nanotechnology-based biochemical assays to study gene expression and function in sorted single cells. The proposed technology will target complex patientderived materials such as peripheral blood and bone marrow. Initial efforts will concentrate on the sorting, enumeration and molecular characterization of four critical molecular targets in lymphocytes. We will develop nanotechnology based assays including nanopore stochastic sensors and fluorescent molecular biosensors to measure protein concentration and kinase activity at the single cell level. The nano-assays will interrogate FoxPS and phospho-ERK concentration as well as kinase activity of cdk4/6 and cdc42all targets of known relevance in cancer biology. The methods described in this proposal will have several immediate clinical applications including the enumeration and characterization of T-regulatory cells, an important T-cell subset that presently cannot be unambiguously identified by flow cytometric means. The technologies developed in specific aims 1-3 of this proposal will allow the tremendous benefits of flow-cytometry to be incorporated into real-time clinical decision making, and will allow for point-of-care testing. Coupled with the nano-analysis capabilities developed in specific aims 4-6, these technologies could be tailored for use in resolving particularly difficult clinical issues in oncology such as determining the expression of a targeted kinase in circulating malignant cells (e.g. Flt3 in AMI); identifying circulating solid tumor cells (e.g. in breast cancer); and identifying kinase-related signaling events in poorly characterized cell types of major biological interest (e.g. hematopoetic stem cells).